Mouse erythrocyte carriers osmotically loaded with methotrexate

The mouse red blood cell (RBC) and red blood cell ghost (RBCG) have been studied as carriers of methotrexate (MTX). When incubated with high concentrations of MTX, RBCs take up significant quantities of it. However, when active loading techniques, such as the slow dialysis and preswell methods, are applied to those cells, up to 15 times more MTX can be entrapped. We have studied factors critical to the incorporation, leakage, and morphology of RBCGs during their loading with MTX by the slow dialysis and preswell methods. Compounds added to the buffers to maintain the ATP content of the cells and osmolarity play functional roles in this process. The fate of the material entrapped within the ghosts after in vivo administration was shown to be capture by the reticuloendothelial system. The pharmacological efficacy of MTX-loaded RBCGs in treating mice bearing hepatoma ascites tumors was demonstrated by increases in average survival time of 28.5-42.8%.     

Immunochemical detection, physicochemical characterization and levels of pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG) in seminal plasma of men

Pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG), the major secretory protein of the human uterine endometrium during the luteal phase of the menstrual cycle and early first trimester of pregnancy, has been detected by immunochemical techniques in seminal plasma. Biochemical analysis and immunoblotting has verified that immunoreactive alpha 2-PEG in seminal plasma exhibits properties identical to those of endometrial alpha 2-PEG, i.e. Concanavalin A-binding dimeric glycoprotein of native Mr 56,000, subunit Mr 28,000, average pI 4.7 and of alpha 2-mobility. Concentration of alpha 2-PEG in seminal plasma was 22.13 +/- 2.82 micrograms/ml (mean +/- s.e.m., n = 110) which compared to 12.02 +/- 1.65 micrograms/ml (mean +/- s.e.m., n = 48) found in amniotic fluid at 11-20 weeks of pregnancy, to 4.29 +/- 1.66 micrograms/ml (mean +/- s.e.m., n = 15) in uterine luminal fluid in women during the luteal phase and to 0.245 +/- 0.025 micrograms/ml (mean +/- s.e.m., n = 10) in sera at 10 weeks of pregnancy. This distribution is very different from that observed for pregnancy-associated placentally-derived serum proteins detected in seminal plasma. The source of alpha 2-PEG in seminal plasma is unknown but is unlikely to be the testis because of the normal levels observed in vasectomized men. In the endometrium alpha 2-PEG synthesis and secretion appears to be related to progesterone-dependent differentiation of the glandular epithelium. Therefore these observations suggest that a different mechanism of regulation of the gene for alpha 2-PEG operates in the male reproductive tract.     

Messenger RNAs of a strongly-expressed late gene of cowpox virus contain 5''-terminal poly(A) sequences.

We have identified and characterized one of the most strongly-expressed genes of cowpox virus (CPV). This is the gene encoding the major protein component of the A-type inclusion bodies produced by this virus. This gene (designated the 160K gene) is transcribed late during the infection. Analyses of its mRNAs showed that these late RNAs, unlike all other characterized late mRNAs of poxviruses, are uniform in length. However, the most remarkable feature of the mRNAs of the 160K gene is the structure of their 5'-termini. Most of these mRNAs have 5'-terminal poly(A) sequences containing 5-21 residues. Furthermore, these 5'-terminal poly(A) sequences are not complementary to the corresponding region of the template strand of the viral DNA. Instead, the nucleotide sequences of the mRNA and the viral DNA diverge at the site of the three As in the sequence 5'-TAAATG-3' containing the gene's initiation codon. Consequently, the poly(A) provides the leader sequences of these mRNAs. These unusual 5'-terminal structures suggest that the late mRNAs of pox-virus genes are generated by a novel process.     

Measurement of serum glycosylated proteins and glycosylated low density lipoprotein fraction in diabetes mellitus

A simple method was developed for estimating serum glycosylated protein levels using gel filtration with Bio-Gel P6 by determining the protein and sugar content in the void volume fraction. The glycosylated protein levels (GSP) correlated well with fasting blood sugar levels and glycosylated albumin level (G-ALB) determined by affinity chromatography with Blue Sepharose CL6B. The glycosylation level of heparin-citrate precipitable fraction of serum which predominantly contained low density lipoprotein (G-LDL) also correlated well with GSP and LDL-cholesterol levels. Significantly different values were obtained for GSP, G-ALB, and G-LDL between normals and diabetics.     

Factors Involved in Hydrolysis of Microcrystalline Cellulose by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus cellulase obtained by the water elution of residual cellulose from the growth medium was compared with the cellulase activity present in culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that water elution released most of the protein bands which adhered to undigested cellulose from the culture medium. The enzyme in the culture supernatant and that eluted from residual cellulose had specific activities for Avicel hydrolysis that were 20- to 40-fold greater than that of Trichoderma reesei cellulase. However, Ca²⁺ and a reducing agent such as dithiothreitol were required for maximum Avicel hydrolysis rates by these A. cellulolyticus enzyme preparations. The effect of these agents on p-nitrophenyl lactopyranoside hydrolysis suggested that they were required by an exoglucanase component. Supernatant enzyme preparations contained large amounts of carbohydrate which was separated from most of the cellulase protein by phenyl-Sepharose chromatography. Removal of this carbohydrate, which interfered with protein fractionations, allowed for an activity stain analysis of the supernatant enzyme.     

The fibrinogen-derived peptide (RGDS) prevents proteolytic degradation of protein kinase C in platelets by inhibiting platelet aggregation

The effects of the fibrinogen-derived tetrapeptide, Arg-Gly-Asp-Ser (RGDS), on platelet activation processes was studied. At concentrations of 100-300 microM, RGDS completely prevented platelet aggregation induced by all the common platelet agonists, 'weak' and 'strong'. In agreement with earlier views on the aggregation-dependency of weak agonist-induced thromboxane synthesis and 5-hydroxytryptamine (5HT) secretion, RGDS (100-300 microM) inhibited these events induced by ADP, adrenaline and low concentrations of thrombin and collagen but not that induced by high concentrations of thrombin and collagen. 5HT secretion induced by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), was also not affected by RGDS, but proteolytic degradation of the translocated membrane-bound enzyme in PMA-treated platelets, due to the actions of the Ca2+-dependent protease (Ca-DP), was completely prevented such that in the presence of RGDS, sustained increases in membrane-bound PKC activity were observed. PMA alone caused only transient increases in membrane-bound PKC. This effect of RGDS was similar to the effect of E64-d, a recently described inhibitor of Ca-DP in platelets, or the effects seen with PMA in unstirred non-aggregating platelets. It is concluded that RGDS inhibits the actions of Ca-DP in platelets via inhibition of aggregation.     

Conformational transitions in cytidine bulge-containing deoxytridecanucleotide duplexes: extra cytidine equilibrates between looped out (low temperature) and stacked (elevated temperature) conformations in solution

High-resolution homonuclear and heteronuclear two-dimensional NMR studies have been carried out on the self-complementary d(C-C-G-C-G-A-A-T-T-C-C-G-G) duplex (designated GCG 13-mer) in aqueous solution. This sequence contains an extra cytidine located between residues G3 and G4 on each strand of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) and correlated (COSY and relay COSY) spectra for the GCG 13-mer duplex in H2O and D2O solution. The extra cytidine at the bulge site (designated CX) results in more pronounced changes in the NOE distance connectivities for the G3-CX-G4 segment centered about the CX residue compared to the C9-C10 segment on the partner strand opposite the CX residue for the GCG 13-mer duplex at 25 degrees C. The cross-peak intensities in the short mixing time NOESY spectrum also establish that all glycosidic torsion angles including that of CX are anti in the GCG 13-mer duplex at 25 degrees C. The observed chemical shift changes for the CX base protons and the G3pCX phosphorus resonance with temperature between 0 and 40 degrees C demonstrate a temperature-dependent conformational equilibrium in the premelting transition region. The NOE and chemical shift parameters establish that the predominant conformation at low temperature (0 degree C) has the extra cytidine looped out of the helix with the flanking G3.C10 and G4.C9 base pairs stacked on each other. These results support conclusions based on earlier one-dimensional NMR studies of extra cytidine containing complementary duplexes in aqueous solution [Morden, K. M., Chu, Y. G., Martin, F. H., & Tinoco, I., Jr. (1983) Biochemistry 22, 5557-5563. Woodson, S. A., & Crothers, D. M. (1987) Biochemistry 26, 904-912]. By contrast, the chemical shift and NOE parameters demonstrate that the conformational equilibrium shifts toward a structure with a stacked extra cytidine on raising the temperature to 40 degrees C prior to the helix-coil melting transition. The most downfield shifted phosphorus resonance in the GCG 13-mer duplex has been assigned to the phosphate in the C2-G3 step, and this observation demonstrates that the perturbation in the phosphodiester backbone extends to regions removed from the (G3-CX-G4).(C9-C10) bulge site.     

Nucleolar organizer region heteromorphism associated with trisomy-21: a risk factor for non-disjunction?

An unusual nucleolar organizer region (double NOR) on chromosome 13 was observed in a Down syndrome child [47, XY, +21, dNOR(13)]. The variant chromosome was inherited from the mother [46, XX, dNOR(13)]. The extra chromosome 21 in the proband was maternal origin. The frequency of NOR chromosome association showed relatively high frequency in the mother and proband as compared to the controls. The result suggest that chromosome variants involving extra copies of NOR may indeed be involved in the meiotic nondisjunction of chromosome-21.     

Induction of aflatoxin biosynthesis inAspergillus parasiticus by ascorbic acid-mediated lipid peroxidation

The effect ofl-ascorbic acid on the biosynthesis of aflatoxin inAspergillus parasiticus was studied. Ascorbic acid at lower concentrations did not inhibit the growth of fungus but markedly induced aflatoxin biosynthesis. At a concentration of 1000 ppm of ascorbic acid, 4.8-fold higher levels of aflatoxin were detected. Copper did not enhance the induction of toxin synthesis by ascorbic acid when added to the growth medium. Ascorbic acid at 1000 ppm was also found to induce aflatoxin synthesis in resting mycelia. Chloroform (1% vol/vol) was found to induce aflatoxin synthesis under similar conditions. Ascorbic acid in the presence of ferrous ion can cause lipid peroxidation, which in turn is responsible for the induction of aflatoxin synthesis. During the induction of aflatoxin synthesis by ascorbic acid, the uptake of carbon source (acetate) was not affected. This observation suggests that on ascorbic acid treatment a precursor or an intermediate of aflatoxin biosynthesis is synthesized in vivo and is responsible for the higher levels of toxin without increasing the uptake of acetate.     

Pyrimidine.pyrimidine base-pair mismatches in DNA. A nuclear magnetic resonance study of T.T pairing at neutral pH and C.C pairing at acidic pH in dodecanucleotide duplexes

Structural features of pyrimidine.pyrimidine mismatches in the interior of oligonucleotide duplexes have been investigated by high resolution two-dimensional proton nuclear magnetic resonance (n.m.r.) spectroscopy. These studies were conducted on the self-complementary d(C-G-C-T-A-G-C-T-T-G-C-G) duplex (designated T.T 12-mer) and the self-complementary d(C-G-C-C-A-G-C-T-C-G-C-G) duplex (designated C.C 12-mer) containing T.T and C.C pairs located at identical positions four base-pairs from either end of the duplex. Proton n.m.r. studies on the T.T 12-mer duplex were undertaken in the neutral pH range, while studies on the C.C 12-mer duplex were recorded at acidic pH. The proton spectra narrowed considerably on lowering the pH below neutrality for the C.C 12-mer duplex. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) data sets have been recorded on the T.T 12-mer and C.C 12-mer duplexes in high salt H2O and D2O solution. The magnitude of the NOE crosspeaks and the directionality of the NOE connectivities demonstrate that both duplexes are right-handed with all bases, including those at the mismatch site, adopting an anti configuration about the glycosidic bond. The observed base and sugar proton chemical shifts suggest structural similarities for the trinucleotide segments centered about the T.T and C.C mismatches. A NOE is detected between the resolved imino protons of T4 and T9 at the mismatch site, consistent with formation of a stacked "wobble" T4(anti).T9(anti) pair in the T.T 12-mer duplex. A comparison of the imino proton chemical shift and NOE data suggests that the imino-carbonyl hydrogen bonds in the wobble T.T mismatch are weaker than the corresponding imino-carbonyl hydrogen bonds in the wobble G.T mismatch. The 4-amino protons of C4 and C9 at the mismatch site in the C.C 12-mer duplex do not exhibit the pattern of hydrogen-bonded and exposed protons separated by approximately 1.5 parts per million characteristic of cytidine amino protons involved in Watson-Crick G.C pairing. The experimental data are insufficient to differentiate between wobble C(anti).C+(anti) and other pairing possibilities for the mismatch in the C.C 12-mer duplex at acidic pH.